Ceramic-containing bioactive inks and printing methods for tissue engineering applications

ABSTRACT

Ink formulations comprising bioactive particles, methods of printing the inks into three-dimensional (3D) structures, and methods of making the inks are provided. Also provided are objects, such as tissue growth scaffolds and artificial bone, made from the inks, methods of forming the objects using 3D printing techniques, and method for growing tissue on the tissue growth scaffolds. The inks comprise a plurality of bioactive ceramic particles, a biocompatible polymer binder, optionally at least one bioactive factor, and a solvent.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/963,348 that was filed Apr. 26, 2018, the entire contents ofwhich are incorporated herein by reference; which is a continuation ofU.S. patent application Ser. No. 14/450,220 that was filed Aug. 2, 2014,the entire contents of which are incorporated herein by reference; whichclaims priority to U.S. provisional patent application No. 61/861,545that was filed Aug. 2, 2013 and U.S. provisional patent application No.61/993,360 that was filed on May 15, 2014; the entire contents of eachof these applications is incorporated herein by reference.

BACKGROUND

Composite scaffolds of hydroxyapatite (HAp) and polycaprolactone (PCL)or polylactic-co-glycolic acid (PLGA) have been developed. However,these composites have been formed utilizing hot-melt three-dimensionalprinting to produce scaffolds composed primarily of PCL or PLGA withembedded HAp. This technique has many drawbacks: 1) hot-melt printingprohibits incorporation of bioactive factors directly into the material;2) the extrusion process relies on the hot melt suspension to beextrudable, which cannot be practically accomplished when the weightfraction of solid HAp particles is greater than approximately 0.5; 3)because the material is primarily PCL, HAp particles are encapsulatedand are not exposed on the surface of the material, meaning that thesurface properties of the material are those of PCL (i.e., smooth andnot amenable to cell adhesion); 4) final objects made from thecomposites are stiff and brittle, preventing reshaping or modificationafter fabrication; and 5) a hot melt suspension cannot be co-printedinto objects along with temperature sensitive materials, such ashydrogels or cell-encapsulated materials.

SUMMARY

Ink formulations comprising bioactive particles, methods of forming theinks into structures, and methods of making the inks are provided. Alsoprovided are porous tissue growth scaffolds made from the inks, methodsof forming the tissue growth scaffolds using 3D printing techniques, andmethods for growing tissue on the tissue growth scaffolds.

One embodiment of an ink comprises bioactive ceramic particles, such ashydroxyapatite particles; a biocompatible polymer binder, such aspolycaprolactone or polylactic-co-glycolic acid; optionally at least onebioactive factor; and a solvent. In some embodiments, the ink comprisesa mixture of the solvents (for example, two or three solvents), as inthe case when a graded solvent is used. The inks may have a highconcentration of bioactive ceramic particles. For example, someembodiments of the inks comprise at least 70 weight percent of thebioactive ceramic, based on the total combined weight of the bioactiveceramic particles and the biocompatible polymer binder. This includesembodiment of the inks that comprise at least 90 weight percent of thebioactive ceramic, based on the total combined weight of the bioactiveceramic particles and the biocompatible polymer binder.

One embodiment of a method of forming an ink comprises dissolving abiocompatible polymer binder in a first solvent and, optionally, mixingone or more bioactive factors into the solvent to form a solution;dispersing bioactive ceramic particles into a second solvent to form adispersion; and mixing the solution and the dispersion to form the ink.

One embodiment of a method of printing a three-dimensional object usingthe inks comprises printing one or more layers of the ink on a substrateand allowing the printed layers to dry.

One embodiment of a porous scaffold comprises a plurality of layersconfigured in a vertical stack, each layer comprising a materialcomprising a plurality of bioactive ceramic particles in a biocompatiblepolymer binder and, optionally, one or more bioactive factors.

One method of growing tissue on the scaffold comprises seeding thescaffold with tissue-forming cells, or cells that are precursors totissue forming cells, and culturing the seeded-scaffold in a cell growthculture medium.

Other principal features and advantages of the invention will becomeapparent to those skilled in the art upon review of the followingdrawings, the detailed description, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments of the invention will hereafter be describedwith reference to the accompanying drawings, wherein like numeralsdenote like elements.

FIG. 1(A). Schematic representation with cross-section ofhydroxyapatite-polycaprolactone (HAPCL) composite fiber. FIG. 1(B)scanning electron microscopy images of HAPCL composite fiber withcross-section (inset).

FIG. 2(A). Schematic illustration of the extrusion-based printingprocesses: single fibers, with thickness d_(l), are deposited d_(s)distance apart. Subsequent layers are deposited l distance (l˜d_(l)) atorientation, θ, with respect to first layer. This process continuesuntil the final structure is complete.

FIG. 2(B). Top view of a scaffold printed via layer-by-layer extrusion.

FIG. 2(C). Side view of a scaffold printed via layer-by-layer extrusion.

FIG. 2(D). Schematic drawing of the top view of a scaffold printed vialayer-by-layer extrusion.

FIG. 3(A). Image of 3D-Printed HAPCL scaffold with differing porearchitecture for 8-layer 30° advancing angle layers. FIG. 3(B). Image of3D-Printed HAPCL scaffold with differing pore architecture for 8-layer90° advancing angle with 250 μm offset every other layer.

FIGS. 4A-D. Scanning electron micrographs of 3D-printed HAPCL scaffoldswith differing pore architecture. In FIG. 4(A), the printed fibers arenon-linear along their longitudinal axes. FIG. 4(C) is a cross-sectionalview of the scaffold of FIG. 4(A). In FIG. 4(B) the fibers in each layerare oriented substantially parallel, while the fibers in adjacent layersare oriented substantially perpendicular. FIG. 4(D) is a cross-sectionalview of the scaffold of FIG. 4(B).

FIG. 5(A). Tensile mechanical testing results for bulk 90 wt. % HAPCLcomposites. FIG. 5(B). Compressive mechanical testing results for bulk90 wt. % HAPLC composites.

FIG. 6(A). Laser scanning fluorescent confocal microscopy live/deadreconstructions of hMSCs 7 days after seeding on 30° HAPCL. FIG. 6(B).Laser scanning fluorescent confocal microscopy live/dead reconstructionsof hMSCs 7 days after seeding on 90° offset HAPCL. Bright Areas=LiveCells.

FIG. 7(A). Scanning electron micrograph of hMSCs 7 days after seeding on30° HAPCL. FIG. 7(B). Scanning electron micrograph of hMSCs 7 days afterseeding on 90° offset HAPCL.

FIG. 8(A). DNA quantification of hMSCs seeded on 3D-bioplotted 30° HAPCLand 90° offset HAPCL. FIG. 8(B) corresponding normalized alkalinephosphatase activity (ALP) 7, 14, and 28 days after seeding.

FIG. 9(A). hMSC expression of osteopontin on 3D-Bioplotted 30° HAPCL and90° offset HAPCL at 7, 14, and 28 days after seeding in simpleproliferation media, not a osteogenic differentiation media. FIG. 9(B).hMSC expression of collagen on 3D-Bioplotted 30° HAPCL and 90° offsetHAPCL at 7, 14, and 28 days after seeding in simple proliferation media,not a osteogenic differentiation media. FIG. 9(C) hMSC expression ofosteocalcin on 3D-Bioplotted 30° HAPCL and 90° offset HAPCL at 7, 14,and 28 days after seeding in simple proliferation media, not aosteogenic differentiation media.

FIG. 10(A). SEM image of collagen synthesis and deposition by hMSCs onHAPCL scaffolds. FIG. 10(B) histological image of collagen synthesis anddeposition by hMSCs on HAPCL scaffolds.

FIG. 11(A). SEM image of hydroxyapatite synthesis and deposition byhMSCs 28 days after seeding. FIG. 11(B) corresponding energy dispersivex-ray spectrum of deposited mineral.

FIG. 12(A). Laser scanning fluorescent confocal microscopy live/deadreconstructions (upper panel) and corresponding cross-sectional SEMimages (lower panel) of hMSCs on 90° offset HAPCL 7 days after seeding.FIG. 12(B) Laser scanning fluorescent confocal microscopy live/deadreconstructions (upper panel) and corresponding cross-sectional SEMimages (lower panel) of hMSCs on 90° offset HAPCL 14 days after seeding.FIG. 12(C) Laser scanning fluorescent confocal microscopy live/deadreconstructions (upper panel) and corresponding cross-sectional SEMimages (lower panel) of hMSCs on 90° offset HAPCL 28 days after seeding.Note: Bright areas=Live cells.

FIG. 13(A). Laser scanning fluorescent confocal microscopy live/deadreconstructions and corresponding cross-sectional SEM images of hMSCs on90° offset HAPLGA 1 day after seeding. FIG. 13(B) Laser scanningfluorescent confocal microscopy live/dead reconstructions andcorresponding cross-sectional SEM images of hMSCs on 90° offset HAPLGA 7days after seeding. FIG. 13(C). Laser scanning fluorescent confocalmicroscopy live/dead reconstructions and corresponding cross-sectionalSEM images of hMSCs on 90° offset HAPLGA 56 days after seeding. Note:Bright areas=Live cells.

FIG. 14(A). SEM micrographs of representative fibers produced from hightemperature-printed (hot melt) 25 vol. % (50 wt. %) HAp+PLGA (25-HT).FIG. 14(B). SEM micrographs of representative fibers produced from roomtemperature-printed 25 vol. % (50 wt. %) HAp+PLGA (25-RT). FIG. 14(C).SEM micrographs of representative fibers produced from roomtemperature-printed dichloromethane (DCM)—only 75 vol. % (90 wt. %)hydroxyapatite HAp+PLGA. FIG. 14(D). SEM micrographs of representativefibers produced from HAPLGA. Scale bars 50 μm.

FIG. 15 . Schematic representation of proposed HAPCL/HAPLGA andelastomer distribution within fibers using single or graded solventmixture as a function of time after extrusion.

FIG. 16 . SEM micrograph of HAPCL fiber microstructures, wherein thefiber was printed from an ink composition comprising a graded solvent.Scale bar 5 μm.

FIG. 17 . Cyclic mechanical testing of a 3D-printed structure composedof the HAPLGA composite.

FIG. 18(A). Schematic representation of ceramic particle and elastomerdistribution in unloaded fiber. FIG. 18(B). Schematic representation ofceramic particle and elastomer distribution in compressed fiber. FIG.18(C). Schematic representation of ceramic particle and elastomerdistribution in stretched fiber. FIG. 18(D). Schematic representation ofceramic particle and elastomer distribution in bent fiber. Arrowsrepresent tensile and compressive loads.

FIG. 19 . Upon unloading, a restoring force opposite in direction to theoriginal tensile, compressive, or bending loads causes the fiber toreturn to its initial morphology.

FIG. 20 . Schematic illustration demonstrating that HAPCL and HAPLGAcomposites will exhibit superposed effects of compression, stretchingand bending when loaded, but will rebound to their original architectureupon unloading over time.

FIG. 21(A). SEM micrograph and photograph inset of HAPLGA hand-tied intoa double micro-knot with inset showing fiber cross-section. FIG. 21(B)SEM micrograph and photograph inset of HAPLGA formed into a twistedfiber cable. Scale bars 100 μm.

FIG. 22 . Measured densities of dry and water saturated HAPCL fiber and(*) densities of dry and water saturated bone tissue from literature.

FIG. 23(A). SEM image of an entire HAPLGA scaffold cross-section, withincision site towards top of image, illustrating tissue infiltration.FIG. 23(B). SEM image of a cross-section of representative blood vesselsfound throughout the HAPLGA scaffold. FIG. 23(C). Magnified view ofsingle HAPLGA strut cross-sections and surrounding tissues. FIG. 23(D).High-magnification image of HAPLGA strut-tissue interfaces and formedcapillaries (arrow).

FIG. 24(A). SEM micrograph of explanted HAPLGA at a first magnification.FIG. 24(B). SEM micrograph of explanted HAPLGA at a secondmagnification. FIG. 24(C). SEM micrograph of explanted HAPLGA at a thirdmagnification.

FIG. 25(A). SEM micrograph of a cross-sectional view of a single HAPLGAstrut within the scaffold surrounded by in grown tissue and activevessels (arrow and circles) from explanted HAPLGA 90° scaffold-tissuesamples removed after 35 days in vivo. FIG. 25(B). SEM micrograph of acut blood vessel which was in the middle of transporting red blood cellsas well as other cells (monocyte, arrows) from explanted HAPLGA 90°scaffold-tissue samples removed after 35 days in vivo. FIG. 25(C). SEMmicrograph of a network of ECM, primarily collagen, from explantedHAPLGA 90° scaffold-tissue samples removed after 35 days in vivo. FIG.25(D). SEM micrograph of an artery-vein complexes (dotted box) in closeproximity to the HAPLGA material from explanted HAPLGA 90°scaffold-tissue samples removed after 35 days in vivo. FIG. 25(E). SEMmicrograph showing upper dotted line=blood vessel, lower dottedline=vein from explanted HAPLGA 90° scaffold-tissue samples removedafter 35 days in vivo.

FIG. 26(A). HAPLGA 12×12 cm mesh sheet comprised of three layers (insetshows close up detail of 3DP pattern). FIG. 26(B). HAPLGA sheets foldedinto a crane. FIG. 26(C). HAPLGA snowflake created through folding atwo-layer 20° HAPLGA sheet and selectively cutting along folds to createa radially symmetric pattern.

FIG. 27 . A skull with spine produced by printing the skull and spineseparately, followed by fusing the spine to the base skull viaapplication of an HAPLGA ink to edges of the contacting regions.

DETAILED DESCRIPTION

Ink formulations comprising bioactive particles, methods of printing theinks into three-dimensional (3D) structures, and methods of making theinks are provided. Also provided are tissue growth scaffolds made fromthe inks, methods of forming the tissue growth scaffolds using 3Dprinting techniques, and methods for growing tissue on the tissue growthscaffolds.

The inks comprise a plurality of bioactive ceramic particles, abiocompatible polymer binder, and at least one solvent. In addition theinks may comprise other additives, including at least one bioactivefactor (e.g., genes, proteins, peptides, growth factors, pharmaceuticalcompounds, antibiotics and the like that facilitate tissue growth by,for example, inducing cell differentiation), and plasticizers.

Unlike other high HAp-content biomaterials, which are brittle, requirehigh temperature processing, and have limited bioactivity, highHAp-content biomaterials printed from the present inks can behyperelastic, and may be quickly fabricated at room temperature intocomplex, implantable structures using an extrusion-based 3D-printingplatform. Structures as small as 1 mm³ or as large as many cm³ can befabricated and manipulated post printing via rolling, folding, cutting,or fusing with other pre-formed structures. The hyperelastic structuresmay be cyclically compressed and return to their net original form afterunloading. These properties may be attributed to the presence ofelastomeric polymer binders, along with a characteristic porousmicrostructure resulting from the specific ink formulation and 3Dprinting process. The microstructure not only permits rigid HApparticles to translate upon mechanical loading and return to theiroriginal position upon unloading, but it can also present a compositionand nano- and micro-porosity biomimetic of natural osseus tissues.

As used herein, the term bioactive ceramic refers to a material which iscapable of promoting the growth of new tissue, such as osteo, chondralor osteochondral tissue. In addition to comprising a bioactive material,the ceramic particles are desirably also relatively stiff, capable ofpromoting cell adhesion, and are osteoinductive, osteoconductive and/orchondrogenically active. Some embodiments of the bioactive ceramicssupport osteogenesis and chondrogenesis under specific differentiationmedia conditions. That is, they can be both chondrogenically andosteogenically active. Hydroxyapatite (HAp) is an example of a suitablebioactive ceramic from which the bioactive ceramic particles can becomprised. HAp is a bioactive ceramic and the native mineral componentof natural bone. HAp has osteoconductive and osteoinductive properties,which provide it with the capacity to induce the growth of new, naturalbone on and around the material, as well as biochemically promoting newbone formation. Calcium phosphates, such as tricalcium phosphate (TCP)are additional examples of bioactive ceramics that can be included inthe ink compositions.

As used herein, the term biocompatible refers to a material that doesnot have a significant negative impact on tissue growth and viability.In addition to comprising a biocompatible material, the polymer bindersare desirably also easy to process, elastic and biodegradable. Thebiocompatible polymer binder may also be a bioactive material. Examplesof suitable polymer binders include biocompatible, bioactive polyesters,such as polylactic acid, polyglycolic acid, polylactic-co-glycolic acid(PLGA) (also referred to as polylactide-co-glycolide (PLG)),polycaprolactone (PCL) or any combination of these. PCL and PLGA aresynthetically derived polyesters. They are elastic, degradable, can beused for cartilaginous tissue regeneration and have been shown tosupport stem cell differentiation down osteogenic and chondrogenicpathways. The biocompatibility of the inks and objects printed from theinks is demonstrated using in vivo and in vitro models in the examplesbelow.

The use of elastic polymer binders, such as PCL and PLGA, promotes therobustness of objects, films and coatings formed from the inkcompositions. In addition, when the ink compositions are extruded, theelastomeric binders provide for the formation of extruded stands thatare continuous, flexible and strong. Moreover, 3D-structures that areextruded or 3D-printed from the ink compositions can adopt theelastomeric properties of the elastic polymer binders. Thus, someembodiments of objects that are formed from the ink compositions havehyperelastic mechanical properties, which allow them to ‘bounce back’ totheir original shape after undergoing loading (e.g., compression ortension). In addition, sheets that are 3D-printed from the inks can berolled, folded and cut.

The hyperelastic structures are comprised of incompressible solidscapable of undergoing large degrees of elastic deformation and thenreturning to their original shape upon unloading. The examplesillustrate hyperelastic 3D-printed objects made with HAPCL or HAPLGAinks. The hyperelastic objects rebounded to their original shape afterbeing pulled, up to ˜40%, or compressed upwards of 55%.

The regenerative capacities of the materials are not only dependent onthe chemical properties of the materials, but are also affected by themechanical properties of the materials. For example, stem cells willrespond to a material's stiffness and behave accordingly. Thus, in thoseembodiments comprising HAp and PCL, HAp induces stem cells to primarilyfollow osteogenic differentiation pathways, while PCL induces stem cellsto primarily follow chondrogenic differentiation pathways.

Because the inks can be formulated and printed at relatively lowtemperatures (e.g., room temperature; ˜23° C.), bioactive factors, suchas proteins, peptides, growth factors and genes, and/or pharmaceuticalcompounds can be added to the ink formulation and, subsequentlyincorporated into structures made from the inks, without undergoingheat-induced degradation. In addition, the low-temperature processingmakes it possible to formulate inks having a high bioactive ceramiccontent. For example, some embodiments of the inks, and the structuresmade therefrom, have a bioactive ceramic content of at least 60 weightpercent (i.e., a weight fraction of 0.6), based on the combined weightof the bioactive ceramic and the biocompatible polymer binder. Thisincludes embodiments having a bioactive ceramic content of at least 70weight percent based on the combined weight of the bioactive ceramic andthe biocompatible polymer binder, at least 80 weight percent based onthe combined weight of the bioactive ceramic and the biocompatiblepolymer binder and at least 90 weight percent based on the combinedweight of the bioactive ceramic and the biocompatible polymer binder.Additionally, the ability to print at relatively low temperaturespermits co-printing alongside temperature sensitive materials such ashydrogels and hydrogels containing living cells. This permitsmulti-material, living bone structures to be printed.

The inks can be made by dissolving a biocompatible polymer binder in afirst solvent and mixing one or more bioactive factors into the solventto form a solution; dispersing bioactive ceramic particles into a secondsolvent to form a dispersion; and mixing the solution and the dispersionto form the ink. The second solvent may be, for example, a gradedsolvent, as illustrated in the example, below. Excess solvent can thenbe evaporated from the resulting formulation to provide a more viscousmixture or “paste”. This entire process can be carried out atlow-temperatures, such as at room temperature—significantly lower thanthose typically used in a hot-melt based process. For example, in someembodiments, the methods of making the inks are carried out at atemperature of no greater than about 35° C. This includes embodiments ofthe methods that are carried out at about room temperature (e.g., in atemperature range from about 22 to about 26° C.).

The inks can be used to form a variety of structures using a variety oftechniques. For example, the inks, including the inks in paste form, canbe cast into a film or coating on a substrate or into a mold to create a3D object. However, the inks are particularly well-suited for use asprintable inks in 3D printing applications directed to the fabricationof bioactive scaffolds. Upon casting or printing, the remaining solventcan be fully evaporated, resulting in a solid structure. Organic solventremoval can be facilitated by washing the printed object in ethanol,followed by washing in sterile water. The resulting structure does nottypically require any post-processing. The resulting composite materialis hyperelastic and comprises a continuous, thin matrix of the binder inwhich the ceramic is dispersed. In those embodiments comprising PCL orPLGA and HAp, which are referred to herein as HAPCL composites andHAPLGA composites, respectively, the macro mechanical properties of theresulting material may be dominated by PCL or PLGA, while the micromechanical and bioactive properties may be dominated by HAp.

Coatings, films, scaffolds and other structures made from the inks maybe bioactive, biodegradable and characterized by a rough surface texturethat promotes cell adhesion, proliferation and activity. In additionthey may be characterized by a large elongation to break. As a result,the structures can be used in a variety of tissue engineeringapplications, including meniscal, cartilage, and subchondral bonereplacement and regeneration (i.e. for osteoarthritis, cartilagedefects, and damaged meniscal tissue); other cartilaginous tissues (i.e.ear, nose, esophagus, trachea); ligament-bone fixation devices forimproving integration and restoring mechanical function after ligamentrepair surgery; craniofacial regenerative implants (e.g., skull plate,nose, cheek bone); support and regeneration of tissue followingcorrective surgeries treating cleft pallet; alveolar ridge support andregeneration immediately following or long after tooth removal; spinefusion and regeneration; regeneration in any long bones, hip bones, orbones in the extremities (i.e. hand, wrist, ankle, foot, toes); drug,gene, or growth factor delivery; and biodegradable implants or coatings.

In addition, the ink compositions are able to bond to previouslydeposited layers or separately printed object parts—including objectparts that are themselves printed using the present 3D ink compositions.Therefore, two or more object parts can be fused together using the 3Dink compositions as a self-adhesive. In these applications, the inkcompositions not only act as an adhesive, but also seamlessly meld theobjects together at the location of deposition. As a result, extremelycomplex or very large 3D objects that could otherwise not be easily 3Dprinted directly can be created by seamlessly fusing parts together withthe same ink composition that comprises the parts themselves.

For tissue engineering applications, the inks are desirably used tofabricate tissue growth scaffolds, which are porous structures thatpermit cell integration, tissue ingrowth, and vascularization. Theporous scaffolds can be printed via layer-by-layer extrusion of an inkthrough a print head of a printer, such as a bioplotter (e.g., EnvisionTEC, GmbH), or through the needle of a syringe. The use of 3D printingfor the fabrication of the scaffolds is advantageous because it providesfor regular geometric patterning of the layers that make up thescaffold, which makes it possible to control and tailor the porosity,pore size and pore interconnectivity of the scaffold. For example, theprinted layers may comprise a plurality of printed fibers. In someembodiments, the fibers in each layer are substantially parallel to oneanother, while the fibers in a given layer are not oriented parallel tothe fibers in other layers. A schematic diagram showing the top view ofsuch an embodiment is provided in FIG. 2D. The printing can be carriedout at relatively low extrusion temperatures, including temperatures inthe range from about room temperature (i.e., ˜23° C.) to about 40° C.

The porous scaffolds can be used as tissue growth scaffolds by seedingthe scaffolds with tissue-forming cells, or cells that are precursors totissue-forming cells, within the pores of the scaffolds. Tissue can begrown by culturing the seeded scaffolds in a cell growth culture medium.Human mesenchymal stem cells, hematopoetic stem cells, embryonic stemcells, and induced pluripotent stem cells are examples of precursors totissue-forming cells. Examples of tissue-forming cells includeosteoblasts, chondrocytes, fibroblasts, endothelial cells, and myocytes.Because bioactive factors can be incorporated directed into the scaffoldas it is fabricated, there is no need to incorporate bioactive factorsinto the culture medium. Thus, in some embodiments, the tissue growth iscarried out in a culture medium that is free of bioactive factors thatpromote the growth of the tissue.

EXAMPLES

The following examples illustrate the use of the present high HApcontent inks to form a 3D-printable biomaterial, Hyperelastic Bone (HB),comprised of 90 wt. % hydroxyapatite (HAp) ceramic particles and 10 wt.% biocompatible elastomer.

Briefly, in vitro studies using human mesenchymal stem cells, describedbelow, reveal that HB is highly supportive of cellular activity. Seededstem cells readily proliferate to quickly coat all available surfacesand fill the inter-scaffold pore volume. HB is also inherentlyosteoinductive, promoting osteogenic differentiation of stem cells,including extracellular matrix (ECM) deposition and de novomineralization without the need for additional osteogenic chemical ormechanical factors. Histological and electron microscopy imaging ofsubcutaneously implanted HB scaffolds in a mouse model, compared tohot-melt 3D-printed HA-polymer scaffolds, reveal that host tissue morereadily integrates within and vascularizes throughout the HB scaffoldswithout any observable host immune response. 3D-printed hyperelasticbone's unique mechanical and biological properties, combined with theease of fabrication, potential for scalability, and low material andprocessing costs make this material system a very promising newosteogenic bone substitute for orthopaedic, dental, and craniofacialtissue regeneration applications.

Example 1

This example describes the room temperature synthesis of a castable and3D-printable HAp-dominant HAPCL composite material comprised of micronor nano-scale HAp particles bound together with a thin, percolatingnetwork of biocompatible, elastic PCL (FIG. 1 ). The resulting materialhas the following properties: rough surface dominated by exposed HApparticles; macro mechanical properties dominated by PCL (elastic, largeelongation to break); micro mechanical properties dominated by HApparticles; biodegradable; osteoinductive; and easy to form into complex,porous scaffold architectures.

Materials and Methods

All processes were performed at room temperature in atmosphere unlessotherwise noted. The desired weight percent (wt. %) of HAp relative toPCL of the final structure was first determined (e.g. 90 wt. % HAp=9 gHAp to 1 g PCL). 9 g HAp (micro or nano-scale) was suspended in amixture of dichloromethane, 2-butoxyethanol, and dibutyl phthalate atrespective mass ratios of 8:2:1. The HAp suspension was then sonicatedfor at least 1 hour. Separately, 1 g of PCL was fully dissolved in 6 gdichloromethane. If bioactive factors were incorporated, these factorswere added in the 6 g dichloromethane prior to addition of PCL. Oncefully dissolved, the PCL solution should be viscous but will still flowunder its own weight. The HA-graded solvent suspension was then added tothe PCL solution, physically mixed for several minutes and sonicated forat least one hour. This process ensured that all HAp particles weredispersed and coated with solubilized PCL and bioactive factors. Ifcasting the material, it should be cast into the mold or container ofinterest at this point. Excess solvent was permitted to evaporateovernight or until the material was dry. If the intended end use is 3Dprinting, excess solvent is evaporated until the HAPCL ink attains aviscosity of approximately 25 Pas. The evaporation rate can be increasedby sonicating the mixture at 40° C.

At this point, the mixture was added to a 3D printer extrusioncartridge. For the purposes of this work, an EnvisionTec Gmbh3D-Biopotter® (Germany) was used. FIG. 16 is an SEM micrograph of HAPCLfiber microstructure. The material was then fashioned into designer,porous 3D objects via layer-by-layer extrusion (shown schematically inFIG. 2A) from a conical 200 μm-diameter polyethylene nozzle at 6.2 barpressure and 4 mm/s speed or from a conical 400 μm polyethylene nozzletip 4 bar pressure and 8 mm/s. Top and side views of printed structuresare shown in FIGS. 2B and 2C, respectively. Other larger nozzlediameters may be used, but only two are described for the purposes ofthis example. The material was extruded onto PTFE coated substrates,which were placed on ice after printing to lift the printed structures.(Many other polymeric and non-polymeric substrates, such as sandpaper,could also be used.) Due to the high vapor pressure of the solvents, andsmall volume of extruded material, the HAPCL strands immediately driedupon deposition onto the substrate. Examples of printed scaffoldstructures are shown in the images in FIG. 3 and SEM micrographs in FIG.4 . FIG. 3A shows an 8-layer 30° advancing angle structure and FIG. 3Bshows an 8-layer 90° advancing angle structure with a 250 μm offsetevery other layer. In FIG. 4 , the spherical objects are HAp particles,and the smooth material between particles is PCL. In FIG. 4A, theprinted fibers are non-linear along their longitudinal axes. FIG. 4C isa cross-sectional view of the scaffold of FIG. 4A. In FIG. 4B the fibersin each layer are oriented substantially parallel, while the fibers inadjacent layers are oriented substantially perpendicular. FIG. 4D is across-sectional view of the scaffold of FIG. 4B. If the scaffold objectsare intended for in vitro use with living cells or in vivo work withanimals or human patients, they are first submerged in sterilizeddeionized water for at least three days to remove organic residues.

Mechanical Characterization:

A 90 wt. % HAp HAPCL solution was cast into PTFE coated petri dishes andpermitted to dry. Flat tensile specimens with 20 cm gauge length and 2.2mm thickness were loaded at an extrusion rate of 1 mm/min. The resultingtensile properties are displayed in FIG. 5A and indicate a tensilemodulus of 15±4.1 MPa and average elongation at break of 40%. Thesevalues are much more similar to PCL than to HA. Compression tests wereconducted on solid, 6 mm-diameter, 3 mm-thick disks. The results areshown in FIG. 5B. The compressive modulus was evaluated at 15% strain tobe 60 MPa.

Incorporation of Bioactive Factors:

To demonstrate that bioactive factors could be successfully incorporatedwithin the material and remain functional during the fabricationprocess, green fluorescent protein (GFP) was added to the PCL solution(5 μg GFP/1 g DCM). The synthesis procedure continued as described aboveand the material was printed into a porous scaffold form and imagedunder black light. Conservation of GFP activity was evidenced by thegreen fluorescence from the GFP incorporated sample.

In Vitro Biocompatibility And Osteogenic Potential:

To ensure that the HAPCL synthesis, fabrication, and washing processleft the material with no harmful organic residues, as well as tomeasure the material's biocompatibility and osteogenic potential, invitro cell studies using human mesenchymal cells (hMSCs) on 3D-printed90 wt. % HAp scaffolds with 200 μm pores were performed. The results ofthese studies at multiple time points are shown in the figures: FIG. 6—Laser scanning fluorescent confocal microscopy live/deadreconstructions of hMSCs 7 days after seeding on 30° offset HAPCL (FIG.6A) and 90° offset HAPCL (FIG. 6B) (Bright Areas=Live Cells); FIG. 7—SEM of hMSCs 7 days after seeding on: (A) 30° HAPCL and (B) 90° offsetHAPCL (hMSCs successfully adhere to and produce extra cellular matrix);FIG. 8 —. (A) DNA quantification of hMSCs seeded on 3D-bioplotted 30°HAPCL and 90° offset HAPCL and (B) corresponding normalized alkalinephosphatase activity (ALP) 7, 14, and 28 days after seeding (cell numberincreases over 4 weeks and ALP activity is significantly higher at 28days after seeding); and FIG. 9 —hMSC expression of osteogenic relevantgenes on 3D-Bioplotted 30° HAPCL and 90° offset HAPCL at 7, 14, and 28days after seeding in simple proliferation media, not a osteogenicdifferentiation media. Expression of osteogenic relevant genes increasessignificantly over the course of 28 days indicating successfulosteogenic differentiation of the MSCs due to interaction with the HAPCLscaffold. Laser scanning fluorescent confocal microscopy images oflive/dead reconstructions and corresponding cross-sectional SEM imagesof hMSCs on 90° offset HAPCL at day 7, day 14 and day 28, are shown inFIGS. 12A, 12B and 12C, respectively.

The utility of the scaffolds is further illustrated by the results shownin FIG. 10 and FIG. 11 . FIG. 10 shows the collagen synthesis anddeposition by hMSCs on HAPCL scaffolds as shown in: (A) SEM; and (B)histological images. hMSCs actively lay down collagen and other extracellular matrix elements in HAPCL. FIG. 11 shows hydroxyapatitesynthesis and deposition by hMSCs 28 days after seeding as shown in: (A)SEM; and (B) corresponding energy dispersive x-ray spectrum of depositedmineral. hMSCs actively synthesize and deposit hydroxyapatite (calciumto phosphate ratio 1.69) in HAPCL. Natural hydroxyapatite found in boneis 1.67. The HAp used in scaffold fabrication is 1.59. Cells in HAPCLproduce HAp that is more natural than the original scaffold. Each ofthese results indicate that stem cells on the HAPCL material wereviable, proliferate, actively produce extracellular matrix, and wereundergoing osteogenic differentiation.

Example 2

This example describes the room temperature synthesis of a castable and3D-printable HAp-dominant HAPLGA composite material comprised of micronor nano-scale HAp particles bound together with a thin, percolatingnetwork of biocompatible, elastic PLGA. The resulting material has thefollowing properties: rough surface dominated by exposed HAp particles;macro mechanical properties dominated by PLGA (elastic, large elongationto break); micro mechanical properties dominated by HAp particles;biodegradable; osteoinductive; and easy to form into complex, porousscaffold architectures. Using in vivo subcutaneous implant testing onmice, this example demonstrates that the resulting printed material isbiocompatible with no evidence of acute or chronic immune response. Thepromotion of vascularization by the material is also demonstrated usingin vivo animal models.

Materials and Methods

All processes were performed at room temperature in atmosphere unlessotherwise noted. The desired weight percent (wt. %) of HAp relative toPLGA of the final structure was first determined (e.g. 90 wt. % HAp=9 gHAp to 1 g PLGA). 9 g HAp (micro or nano-scale) was suspended in amixture of dichloromethane, 2-butoxyethanol, and dibutyl phthalate atrespective mass ratios of 8:2:1. The HAp suspension was then sonicatedfor at least 1 hour. Separately, 1 g of PLGA was fully dissolved in 6 gdichloromethane. If bioactive factors were incorporated, these factorswere first dissolved in the 6 g dichloromethane prior to addition ofPCL. Once fully dissolved, the PLGA solution should be viscous but willstill flow under its own weight. The HA-graded solvent suspension wasthen added to the PLGA solution, physically mixed for several minutesand sonicated for at least one hour. This process ensured that all HApparticles were dispersed and coated with solubilized PLGA and bioactivefactors. If casting the material, it should be cast into the mold orcontainer of interest at this point. Excess solvent was permitted toevaporate overnight or until the material was dry. If the intended enduse is 3D printing, excess solvent is evaporated until the HAPLGA inkattains a viscosity of approximately 25 Pas. The evaporation rate can beincreased by sonicating the mixture at 40° C. At this point, the mixturewas added to a 3D printer extrusion cartridge. For the purposes of thiswork, an EnvisionTec Gmbh 3D-Biopotter® (Germany) was used. The materialwas then fashioned into designer, porous 3D objects via layer-by-layerextrusion from a conical 200 μm-diameter polyethylene nozzle at 6.2 barpressure and 4 mm/s speed or from a conical 400 μm polyethylene nozzletip 4 bar pressure and 8 mm/s. Other larger nozzle diameters may beused, but only two are described for the purposes of this example. Thematerial was extruded onto PTFE coated substrates, which were placed onice after printing to lift the printed structures. Due to the high vaporpressure of the solvents, and small volume of extruded material, theHAPLGA strands immediately dried upon deposition onto the substrate.

Residual solvents and residues were removed from the 3D-printed HAPLGAcomposites following washes in 70% ethanol followed by several rinses insterile water or phosphate buffered saline. Thermogravimetric analysis(TGA) of the HAPLGA scaffolds pre- and post-washing was used todetermine if residual solvents were completely removed from the materialprior to application in an in vitro or in vivo environment. As-printedscaffolds contain as much as 18 wt. % solvent. A 1 hour rinse in DIwater left approximately 9 wt. % solvents. If rinsed in 70% ethanol(EtOH) followed by a water rinse, all residual solvents were removedfrom the material. Note: that also verified that HAPLGA was 90 wt. %HAp.

For comparison, fibers were also made via hot melt printing an inkcomposition comprising 25 vol. % (50 wt. %) HAp+75 vol. % (50 wt. %)PLGA. Fibers made from this ink composition are referred to herein as25-HT fibers. The ink for the 25-HT fibers was formulated by mixing PLGAand HAp powder (1:1 by weight). This mixture was then loading into aBioplotter high temperature cartridge, which was heated to 200° C. After1 hour, the cartridge was maintained at 200° C. and the material was3D-printed via extrusion. Solidification of extruded 25-HT fibersoccurred due to melted polymer being exposed to room temperature andsolidifying. The 25-HT fibers represent a common class of bioactiveceramic/polymer composites made by mixing a polymer binder and ceramicpowders together, melting the polymer binder, and 3D printing viaextrusion. Additional comparative fibers were made using an inkcomposition comprising 25 vol. % (50 wt. %) HAp++75 vol. % (50 wt. %)PLGA, which was processed into a room temperature printable ink in thesame manner as the HAPLGA ink, except that the HAp and PLGA werecombined is a ratio of 1:1 by weight, rather than a ratio of 9:1 byweight. Fibers made from this ink composition are referred to herein as25-RT fibers. Finally, fibers were made from an ink comprising 75 vol. %(90 wt. %) HAp and 25 vol. % (10 wt. %) PLGA. This ink was formulatedusing the same procedure used to formulate the HAPLGA ink, except that asingle solvent, DCM, was used rather than the mixed DCM,2-butoxyethanol, and dibutylphthalate solvent used to formulate theHAPLGA ink. Fibers made from this ink composition are referred to hereinas DCM-only fibers. FIG. 14 shows SEM micrographs of the fibers: FIG.14A—25-HT fiber; FIG. 14B—25-RT fiber; and FIG. 14C—DCM-only fiber. FIG.14D shows the HAPLGA fiber, respectively. FIG. 16 shows a magnifiedportion of the HAPLGA fiber of FIG. 14D.

FIG. 15 shows a proposed mechanism that allows the inks to be 3D-printedand also formed into characteristic microstructures that are responsiblefor the bioactive and mechanical properties of the HAPLGA composites.

Characterization:

The 3D-printed HAPLGA structures were cyclically compressed more than40% and returned to net original shape and strength upon unloading. Theresults of the testing are shown in FIG. 17 . The hysteresis in thecurve in FIG. 17 illustrates that upon compression and release, theHAPLGA composite material bounced back (demonstrating hyperelasticity)and was able to be loaded again. Ten cycles were using in this test.However, the hysteresis curves are so close to overlapping that the datafor all 10 cycles cannot be individually distinguished.

FIG. 18 provides a schematic representation of the ceramic particle andelastomer distribution in: (A) an unloaded; (B) a compressed; (C) astretched; and (D) a bent fiber. Arrows in the figures represent tensileand compressive loads. Upon unloading, a restoring force opposite indirection to the original tensile, compressive, or bending loads causesthe fiber to return to its initial morphology, as shown in FIG. 19 . Asillustrated schematically in FIG. 20 , the HAPLGA will exhibitsuperposed effects of compression, stretching and bending when loaded,but will rebound to their original architecture upon unloading overtime.

FIG. 21A is an SEM micrograph of HAPLGA fibers hand-tied into a doublemicro-knot (the inset shows the fiber cross-section) and FIG. 21B is anSEM micrograph of HAPLGA fibers formed into a twisted fiber cable. Scalebars 100 μm.

The densities of dry and water saturated HAPCL fibers and (*) densitiesof dry and water saturated bone tissue from literature were alsomeasured and the results are shown in FIG. 22 .

In Vitro Biocompatibility and Osteogenic Potential:

To ensure that the HAPLGA synthesis, fabrication, and washing processleft the material with no harmful organic residues, as well as tomeasure the material's biocompatibility and osteogenic potential, invitro cell studies using human mesenchymal cells (hMSCs) on 3D-printed90 wt. % HAp scaffolds with 200 μm pores were performed. Laser scanningfluorescent confocal microscopy images of live/dead reconstructions atday 1, day 7 and day 56, are shown in FIGS. 13A, 13B and 13C,respectively.

In Vivo Biocompatibility:

The 90° HAPLGA scaffolds were implanted subcutaneously (under the backskin) of BALB/c mice. Scaffolds were removed 7 or 35 days afterimplantation and observed using histology and SEM. No evidence of acuteor chronic immune responses was found at either time point. It was alsofound that HAPLGA became highly vascularized and well integrated withthe surrounding tissues. This confirmed the biocompatibility of HAPLGAas well as the potential for healthy tissue integration.

FIG. 23 shows SEM images of: (A) the entire scaffold cross-section, withincision site towards top of image; (B) a cross-section ofrepresentative blood vessels found throughout HAPLGA scaffold; (C) amagnified view of single HAPLGA strut cross-sections and surroundingtissues; and (D) a high-magnification image of HAPLGA strut-tissueinterfaces and formed capillaries (arrow).

FIG. 24A-24C show SEM micrographs of explanted HAPLGA tissue growthscaffolds at different magnifications for samples removed after 35 daysin vivo. The in-grown tissue forms intimate contact with the 3D-printedstruts throughout the scaffold volume. The material of FIG. 24A is openand contains many more small and large vessels. The dotted circles inthe magnified image shown in FIG. 24B are healthy red blood cells. Alarger image of healthy red blood cells is provided in FIG. 24C.

FIG. 25 shows the SEM micrographs of explanted HAPLGA 90°scaffold-tissue samples removed after 35 days in vivo. FIG. 25A is across-sectional view of a single HAPLGA strut within the scaffoldsurrounded by in-grown tissue and active vessels (arrow and circles).The vasculature was active up until the point the scaffold-tissue samplewas fixed and cut in half, as can be seen by the cut blood vessel whichwas in the middle of transporting red blood cells as well as other cells(FIG. 25B, monocyte, arrows). A network of ECM, primarily collagen, wasobserved to comprise the majority of tissue within the scaffold volume(FIG. 25C). Artery-vein complexes (FIG. 25D, dotted box) were alsoobserved in close proximity to the HAPLGA material (FIG. 25E, upperdotted line=blood vessel, lower dotted line=vein).

Mechanical Flexibility:

The mechanical flexibility of sheets printed from the HAPLGA-based inkcompositions was demonstrated by rolling, folding and cutting thesheets. FIG. 26A shows a 12×12 cm mesh sheet comprised of three layersprinted from an HAPLGA ink (inset shows close up detail of 3DP pattern).FIG. 26B shows that the printed HAPLGA sheets can be folded to create acomplex structure such as a crane. FIG. 26C is a snowflake createdthrough folding a two-layer 20° HAPLGA sheet and selectively cuttingalong folds to create a radially symmetric pattern.

Use of Inks as Adhesives:

To illustrate the ability of the ink compositions to act as adhesives inbonding pre-fabricated parts into a single object, a skull with a spinewas produced by printing the skull and spine separately using an HAPLGAink, followed by fusing the spine to the base skull via application ofan HAPLGA ink to edges of the contacting regions. The skull in this casewas approximately 340 printed layers thick (not including the spine).The spine itself was printed in two separate sections (halves along itslength). The spine halves were fused together using the HAPLGA ink,dispensed via syringe by hand, along the seams. The spine was then fusedto the skull using the HAPLGA ink, by dispensing the ink via syringe byhand to the base of the skull and contacting the spine to the ink.Finally, the jaw was printed separately and fused to the skull using theHAPLGA ink as an adhesive. The resulting skull and spine are shown inFIG. 27 . The fabrication of the skull and spine illustrates thescalability of the printing to produce a large complex object comprisinghundreds of printed layers and also illustrates the ability of the inkcompositions to seamlessly fuse independently printed parts.

The word “illustrative” is used herein to mean serving as an example,instance, or illustration. Any aspect or design described herein as“illustrative” is not necessarily to be construed as preferred oradvantageous over other aspects or designs. Further, for the purposes ofthis disclosure and unless otherwise specified, “a” or “an” means “oneor more”.

The foregoing description of illustrative embodiments of the inventionhas been presented for purposes of illustration and of description. Itis not intended to be exhaustive or to limit the invention to theprecise form disclosed, and modifications and variations are possible inlight of the above teachings or may be acquired from practice of theinvention. The embodiments were chosen and described in order to explainthe principles of the invention and as practical applications of theinvention to enable one skilled in the art to utilize the invention invarious embodiments and with various modifications as suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto and theirequivalents.

What is claimed is: 1.-20. (canceled)
 21. A solvent-based inkcomprising: bioactive ceramic particles; a biocompatible polymer binder;and at least one solvent, wherein the ink comprises at least 70 weightpercent of the bioactive ceramic particles, based on the total combinedweight of the bioactive ceramic particles and the biocompatible polymerbinder and the ink is extruded at room temperature with the solventevaporated upon extruding, forming one or more porous fibers comprisinga microstructure comprising a continuous matrix of the biocompatiblepolymer binder with intra fiber pores and the bioactive ceramicparticles dispersed throughout the microstructure.
 22. The ink of claim21, further comprising at least one bioactive factor.
 23. The ink ofclaim 21, wherein the biocompatible polymer binder is a degradablepolyester and the bioactive ceramic particles are comprised ofhydroxyapatite, tricalcium phosphate, or a combinations thereof.
 24. Theink of claim 21, wherein the bioactive ceramic particles arehydroxyapatite particles and the ink comprises at least 80 weightpercent of the hydroxyapatite particles, based on the total combinedweight of the hydroxyapatite particles and the polycaprolactone.
 25. Theink of claim 21, wherein the biocompatible polymer comprisespolylactic-co-glycolic acid, polylactide-co-glycolide, orpolycaprolactone.
 26. The ink of claim 25, wherein the bioactive ceramicparticles are hydroxyapatite particles and the ink comprises at least 80weight percent of the hydroxyapatite particles, based on the totalcombined weight of the hydroxyapatite particles and thepolylactic-co-glycolic acid, polylactide-co-glycolide, orpolycaprolactone.
 27. The ink of claim 22, wherein the at least onebioactive factor selected from the group consisting of proteins,peptides, growth factors, genes, pharmaceutical compounds, antibioticsand combinations thereof.
 28. The ink of claim 21, comprising at least90 weight percent of the bioactive ceramic particles, based on the totalcombined weight of the bioactive ceramic particles and the biocompatiblepolymer binder.
 29. The ink of claim 21, further comprising at least onebioactive factor without heat induced degradation.
 30. The ink of claim21, comprising at least 80 weight percent of the bioactive ceramicparticles, based on the total combined weight of the bioactive ceramicparticles and the biocompatible polymer binder.
 31. The ink of claim 21,wherein the ink is characterized in that it can be extruded as acontinuous flexible fiber through a 3D printer nozzle having a diameterin the range from 100 μm to 400 μm using a pressure in the range from6.2 bar to 4 bar and a printing speed in the range from 4 mm/s to 8 mm/sat a temperature in the range from 22° C. to 40° C.
 32. The ink of claim21, wherein the solvent comprises a high volatility solvent, a secondarysurfactant solvent having a vapor pressure lower than the highvolatility solvent, and a plasticizer.
 33. The ink of claim 21, whereinthe solvent comprises a mixture of dichloromethane, 2-butoxyethanol, anddibutylphthalate.
 34. The ink of claim 21, wherein the bioactive ceramicparticles include bioactive ceramic particles having diameters in therange from 5 μm to 20 μm.
 35. The ink of claim 21, characterized in thatit can be extruded as a continuous elastic fiber through a nozzle,wherein the elastic fiber can be elastically deformed by a compressiveload and then return to its original shape upon unloading.
 36. The inkof claim 21, wherein the ink is characterized in that it can be extrudedas a continuous flexible fiber through a 3D printer nozzle having adiameter equal to or greater than 100 μm.
 37. The ink of claim 21,wherein the ink is characterized in that it can be extruded as acontinuous flexible fiber having a diameter of about 200 μm through a 3Dprinter nozzle.
 38. The ink of claim 21, wherein the ink ischaracterized in that it can be extruded as a continuous flexible fiberthrough a 3D printer nozzle having a diameter equal to or greater than400 μm.
 39. The ink of claim 21, wherein a diameter of the intra fiberpores are in a range from about 1 μm to about 10 μm.
 40. The ink ofclaim 21, wherein the biocompatible polymer binder comprises anelastomeric polymer.
 41. The ink of claim 21, wherein the biocompatiblepolymer binder comprises a biodegradable polymer.
 42. A solvent-basedink comprising: bioactive ceramic particles; a biocompatible polymerbinder; and at least one solvent, wherein the ink comprises at least 70weight percent of the bioactive ceramic particles, based on the totalcombined weight of the bioactive ceramic particles and the biocompatiblepolymer binder, the biocompatible polymer binder makes up from 10 weightpercent to 30 weight percent of the combined weight of the bioactiveceramic particles and the biocompatible polymer binder, and the ink isextruded at room temperature with the solvent evaporated upon extruding,forming one or more porous fibers comprising a microstructure comprisinga continuous matrix of the biocompatible polymer binder with intra fiberpores and the bioactive ceramic particles dispersed throughout themicrostructure.